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1.
Energetically-coupled processes (electron flow, proton uptake and correlated pH gradient) were investigated on envelope-free chloroplasts of lettuce suspended in 1H2O or 2H2O media. Study of the light-intensity and temperature dependencies of these phenomena led to the following observations: 1. At neutral pH, 2H2O diminishes the transmembrane H+ gradient in strong light (chain Photosystem II + Photosystem I) but not in low light; the total H+ uptake is increased at all light intensities: the buffering capacity of the inner compartment is increased in heavy water, possibly through enhancement of interactions between membranous titrable groups and the aqueous phase. 2. 2H2O does not affect the photochemical events of the redox chain, whatever the electron pathway (PSII, PSI or PSII + PSI): only thermal steps are inhibited. The diminution of the apparent quantum yield, sometimes observed, may be ascribed to the dual site of action of the artificial redox carrier (ferricyanide) then used. 3. 2H2O does not modify the activation energy of the limiting step of the electron flow (PSII + PSI) in uncoupled (44 vs. 47 kJ · mol?1) or — but less clearly — in coupled, i.e., ‘basal’, state (55 vs. 59 kJ · mol?1). 2H2O does not either change the temperature of the phase transition of the membrane (17°C) for the uncoupled flow. However, a low-temperature transition, observed only for the coupled chain, is slightly increased by 2H2O; this thermal transition is attributed to the freezing of some bound water near the plastoquinone pool. 4. Δp2H is smaller than Δp1H at all temperatures (PSII + PSI chain). ΔpH is quasi-constant from 0°C to 10°C, then decreases when temperature rises. 2H2O does not change the activation energy of the dark passive H+ efflux, which is almost twice that of the coupled electron flow. The phase transition at low temperature suggests that the proton efflux occurs via two parallel pathways, one temperature-dependent and the other temperature-independent. Except for the increase of the internal buffering capacity, the effects of 2H2O on the membrane conformation seem limited, as shown by the unchanged activation energies of the electron flow and of the H+ leakage. The null activation energy observed at low temperature emphasizes the role of the bound water in these processes; however, the different effects of 2H2O on the transition temperatures indicate that this bound water has different properties when associated with the translocation sites or with the H+ leakage ones. This ‘microcompartmentation’ of the membranes is consistent with the concept of lateral pH heterogeneity we have previously suggested (de Kouchkovsky, Y., and Haraux, F. (1981) Biochem. Biophys. Res. Commun. 99, 205–212). The theoretical computations and the experimental results suggest that in the steady state, the internal pH would be several tenths of a ‘unit’ lower near the plastoquinones than near the H+ efflux sites (coupling factors); this difference would be increased when 2H+ replaces 1H+, owing to the lower mobility of the deuteron. It is concluded that local, and not average, pH (and ΔpH) should be considered for the understanding of the energy transduction processes.  相似文献   

2.
《BBA》2018,1859(10):1067-1074
In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase (gH+) than the mature leaves. Furthermore, gH+ was significantly and positively correlated with CO2 assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that gH+ was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in gH+. In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO2 assimilation and photoprotection during leaf maturation.  相似文献   

3.
The effect of bicarbonate ions on induction of Mg2+-ATPase activity, on the N-ethylmaleimide inhibition of phosphorylation and on energy-dependent adenine nucleotide exchange has been examined with pea seedling chloroplasts. Incubation of chloroplasts with N-ethylmaleimide in the presence of 15 millimolar bicarbonate in the light results in enhanced inhibition of ATP synthesis when the preillumination pH is maintained between 7.0 and 7.5. Bicarbonate also enhances Mg2+-ATPase activity when it is included in the light-triggering stage at pH 7.0. The conditions (medium pH, bicarbonate concentration, etc.) for demonstrating the bicarbonate-induced enhancement of the N-ethylmaleimide inhibition and ATPase activity are similar to those required for the direct effect of bicarbonate on phosphorylation. Bicarbonate, under the same conditions, does not affect adenine nucleotide exchange (binding or release). It is concluded that the stimulatory effect of bicarbonate on ATP synthesis may be related to its ability to alter directly the conformation of the chloroplast coupling factor under conditions (suboptimal pH) where the enzyme shows minimal activity.  相似文献   

4.
Initial and steady state rates of proton transport at low light intensity have been measured and compared with steady state rates of electron transport in the pH range of 6.0–7.6 in envelope-free spinach chloroplasts. At pH 6–7, the H+/e- values computed using the initial rate of proton transport varied with light intensity, from a value of 2 at low light to almost 5 at high light. In contrast, the H+/e- values computed using the steady state rate of proton transport did not show a dependence on light intensity, having a constant value of 1.7±0.2. Likewise, at pH 7.6, the H+/e- ratio, computed using either the initial or steady state rates of proton transport did not vary with light intensity but was constant at H+/e-=1.7±0.1. Analysis of the light dependence of electron and proton transport allowed determination of (a) the quantam requirements of transport, (b) the rates of transport at light saturation, and (c) H+/e- ratios for initial and steady state proton transport. Extrapolating the initial proton transport to zero light, we found that both H+/photon and H+/e- values were not strongly dependent on pH, approaching a near constant value of 2.0. Using the initial rate of proton transport extrapolated to saturating light intensity we found the H+/e- ratio to be strongly pH-dependent. We suggest that internal pH controls electron transport at high light intensities. The true stoichiometry is reflected only in measurements taken at low light using the initial proton transport data. Our findings and interpretation reconcile some conflicting data in the literature regarding the pH-dependence of the H+/e- ratio and support the concept that internal pH controls noncyclic electron transport.Abbreviations Bicine N, N-bis [2-hydroxyethyl] glycine - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethansulfonic acid - MES 2-(N-morpholino) ethanesulfonic acid  相似文献   

5.
Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout = 7.8, we found that pHin ≈ 5.4-5.7 in the state of photosynthetic control, and pHin ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n ≥ 4-4.7.  相似文献   

6.
The primary stable products of photosynthetic electron flow are NADPH and ATP. Stoichiometry of their production depends on the ratio of protons pumped across the thylakoid membrane to electrons passed through the electron transport pathway (H+/e ratio). Flexible requirements of the ATP/NADPH ratio by various assimilatory reactions in chloroplasts must be fulfilled by the H+/e ratio during the electron flow. In addition to the well-known role of ΔpH during ATP synthesis, ΔpH also functions as a trigger of the down-regulation of photosystem II (PSII) photochemistry. Excessive light energy is safely dissipated as heat by this regulatory process to suppress the generation of toxic reactive oxygen species. Thus, regulation of the H+/e ratio may function in the photoprotection, as well as in the regulation of the ATP/NADPH production ratio. It has long been the consensus that the H+/e ratio can be controlled by regulating the proton-transporting Q-cycle in the cytochrome b 6 f complex and by the cyclic electron flow around photosystem I (PSI). Despite the possible physiological importance and the long history of interest, the molecular identity of Q-cycle regulation and the cyclic electron flow around PSI have been remained unclear. The recent improvements in research tools, including the genetic approach using chlorophyll fluorescence imaging and establishment of the chloroplast transformation technique, are providing new insights into classical topics. In this review, we focus on regulation of the H+/e ratio especially from the view of photosynthetic regulation. Received: August 2, 2001 / Accepted: October 1, 2001  相似文献   

7.
《BBA》1986,850(1):90-96
Broken chloroplasts activated by preillumination in the presence of dithiothreitol were supplied with phosphate and with a limited concentration of ADP. On re-illumination, ATP was formed until a steady state was attained. If after reaching the steady state light intensity was reduced to 20–50 W · m−2, net ATP hydrolysis took place, but after some time in weak light the level of ATP re-increased. Similarly, a drop of transmembrane ΔpH followed by a slow recovery was observed. Further data indicate that the reversible changes of ATP level and ΔpH are the result of partial uncoupling induced by ATP during the preceding strong light period and of restoration of coupling within a few minutes in weak light. Since similar changes of endogenous ATP level were found when intact chloroplasts were subjected to a strong-light/weak-light transition, it is proposed that ATP-induced partial uncoupling may play a role in regulation of photosynthetic energy conservation as a means to dissipate abundant transmembrane electrochemical energy and to permit flexibility of the stoichiometry of ATP-to-NADPH production.  相似文献   

8.
Cornic G  Bukhov NG  Wiese C  Bligny R  Heber U 《Planta》2000,210(3):468-477
The role of cyclic electron transport has been re-examined in leaves of C3 plants because the bioenergetics of chloroplasts (H+/e = 3 in the presence of a Q-cycle; H+/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C3 photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large increase in the number of electrons available to reduce P700+ in the dark. At low CO2 and O2 molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, Fv/Fm) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in Fv/Fm brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO2-dependent O2 evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease Fv/Fm. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H+/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. Received: 23 February 1999 / Accepted: 19 August 1999  相似文献   

9.
《FEBS letters》1985,187(2):257-260
The effect of gramicidin on the phosphorylation rate, electron flow and light-induced H2+ uptake (ΔH+) in chloroplasts with methyl viologen (MV) or phenazine methosulfate (PMS) added has been studied. In the presence of MV low concentrations of gramicidin (~10−9 M) were shown to inhibit the phosphorylation rate up to 80–90% of the initial value, without changing either the electron transport rate or ΔH+ value. Two components of phosphorylation have been identified in the presence of PMS - the first is DCMU-sensitive, which was suppressed by low gramicidin concentrations (< 10−9 M) and the second - DCMU-insensitive component, which was suppressed by high gramicidin concentrations (~10−7 M) exclusively, the high gramicidin concentration inducing the ΔH+ value fo fall.  相似文献   

10.
The dependence of the light-induced H+ gradient in chloroplasts (ΔpH) on external pH was examined using the distribution of aniline, an amine of low pKa. ΔpH was essentially independent of pH over the range of 7–8. It was previously reported that ΔpH, determined from the distribution of relatively polar amines of high pKa, decreased as the pH was lowered below 8. It is suggested that, in the case of amines of high pKa, ΔpH values determined at low external pH values are too low because the permeability of chloroplasts to the amine cation relative to that of the unprotonated form may be significant.  相似文献   

11.
Over‐reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H+ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over‐reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)‐treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over‐reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ‐subunit of chloroplastic CF0CF1‐ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H+ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF0CF1‐ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild‐type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF0CF1‐ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H+ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF0CF1‐ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.  相似文献   

12.
Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 μM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H+). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H+ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.  相似文献   

13.
The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K+ and high levels of Na+, Mg2+ and Ca2+. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H+ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H+, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.  相似文献   

14.
Photosynthesis, stroma-pH, and internal K+ and Cl concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K+, H+, Cl) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K+ or added Tetraphenylphosphonium+) and excludes anions (Cl) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium+ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K+ or Na+ and the release of protons all become very small. Light-induced K+ uptake is not abolished by valinomycin suggesting that the K+ uptake is not primarily active. Intact chloroplasts contain higher K+ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO2 fixation at high K+ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO2 fixation in intact chloroplasts.

  相似文献   

15.
Thomas Graan  Donald R. Ort   《BBA》1982,682(3):395-403
Two saturating single-turnover flashes spaced 100 ms apart are sufficient to achieve ATP formation in isolated chloroplast thylakoids. Two turnovers of the electron carriers result in the accumulation of about 7 nmol H+ / mg chlorophyll. Under the same conditions (i.e., ΔGATP = 38 kJ/mol) a solitary flash is inadequate to produce ATP. The electron flux from the third or any subsequent flash is coupled to ATP formation as efficiently as is observed in continuous light (i.e., ) and produces 0.8 molecules of ATP per coupling factor on each turnover. The yield of ATP per flash increases with declining temperature being largest near 4°C, the lowest value tested. The number of H+ accumulated per flash is independent of temperature so the greater yields of ATP near 4°C indicate that fewer H+ are existing the membrane via nonproductive pathways. The yield of ATP per flash near 4°C is largely independent of flash frequency between 1 and 30 Hz. When the formation of an electrical potential difference is prevented by adequate amounts of valinomycin and potassium the accumulated effects of about eight flashes are required before ATP formation is achieved (i.e., about 26 nmol H+/mg chlorophyll), indicating an average ΔpH/flash in excess of 0.3 units. In the presence of the exchange carrier nigericin, the electrical component of the driving force for ATP formation is enhanced at the expense of the ΔpH. In this case, ATP formation is efficiently coupled to electron flux only at flash frequencies rapid enough to allow a summation of the electrical field. These results clearly demonstrate that any processes which are prerequisites for ATP synthesis (i.e., activation of coupling factor or generation of Δp) are fulfilled by a remarkably small number of charge separations.  相似文献   

16.
U. Heber  M.R. Kirk 《BBA》1975,376(1):136-150
Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP2 e ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 11 ratio, was about 4 both at pH 7.6 and at 7.0. ATP2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2.Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP2e ratios were calculated to be between 1.1 and 1.4.Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg?1 chlorophyll · h?1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP2e ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials [ATP][ADP] [Pi]. Flexible coupling implies, that ATP2e ratios should decrease with increasing phosphorylation potentials inside the chloroplasts.  相似文献   

17.
18.
Huber SC  Maury W 《Plant physiology》1980,65(2):350-354
Exogenous Mg2+ (2 millimolar) altered the stromal pH of intact spinach chloroplasts. Without added KCl in the medium, Mg2+ decreased the stromal pH in the light by approximately 0.3 pH unit. External KCl (25 millimolar) largely prevented the acidification caused by Mg2+. Effects on the stromal pH were not caused by changes in H+ pumping across the thylakoid membrane because Mg2+ had no effect on the light-induced quenching of atebrin fluorescence by intact chloroplasts. However, Mg2+ affected H+ fluxes across the envelope. Addition of Mg2+ to intact chloroplasts in the dark caused a significant acidification of the medium that was dependent on the presence of K+.  相似文献   

19.
Thomas Graan  Donald R. Ort 《BBA》1981,637(3):447-456
Full development of the capacity for ATP formation in isolated thylakoid membranes coincides with the beginning of illumination. Indeed, the yield of ATP per ms of illumination is about twice as great during the first 15 ms of high-intensity illumination as it is thereafter. The presence of valinomycin and K+ prevents the formation of a membrane potential (as indicated by the obliteration of most of the change in absorbance at 518 nm) and at the same time delays the formation of the capacity for ATP synthesis for many milliseconds. Presumably, phosphorylation is initially dependent on a rapidly formed membrane potential, whereas after a delay a ΔpH sufficient to drive ATP formation forms. The actual duration of this delay depends on the phosphoryl group transfer potential (i.e., ΔGATP) of the ATP-synthesizing reaction. If the delay in the presence of valinomycin and K+ represents the time required to develop a ΔpH capable of driving phosphorylation by itself, then the effect of ΔGATP on the duration of the delay suggests that the onset of phosphorylation is determined by the magnitude of the electrochemical potential of protons and not by factors affecting the activation of the coupling factor enzyme. The initial ATP formation, which is almost entirely dependent on the electrical potential, should not be affected by the electrically neutral exchange of cations catalyzed by nigericin. When the external pH is 7.0 this seems to be true, since the ATP synthesis which is initially sensitive to valinomycin and K+ is largely insensitive to nigericin and K+. However, when the external pH is 8.0 the response to nigericin is exactly the opposite and the ATP formation which is sensitive to valinomycin is also abolished by nigericin. These data suggest that there may be either an energetic requirement for both a ΔpH and membrane potential at alkaline pH or a non-energetic requirement for a minimum proton activity in the initiation of phosphorylation.  相似文献   

20.
The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H+/e ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m–2) (strong light) the H+/e ratio was 3. At low intensity (0.9 W · m–2) (weak light) the H+/e ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H+/e ratio due to underestimation of the initial rate of H+ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.  相似文献   

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